This study attempted to recover starch debranching enzyme, isoamylase, from the fermentation broths by using an aqueous two-phase system. A series of poly(ethylene glycol) (PEG) combined with different salt systems were used to prepare the partition systems. In these experiments, the partition coefficient of isoamylase for the PEG1500(6%)/(NH4)(2)SO4(15%) system could be up to 40 when a crude enzyme solution with an isoamylase activity of 3386 U/mL was used, and the enzyme activity was 5.0-fold concentrated in the top phase as compared to the crude enzyme solution. Based on the recovery and concentration fold of isoamylase, a pH around 4 similar to 5 was found to be the optimal condition for this system. The polymer design and combination of salts could be manipulated to achieve different volume ratios between the top and the bottom phases, and could also achieve the purpose of enzyme concentration. For the PEG1500(6%)/(NH4)(2)SO4(15%) system, experimental results indicated a 6.0-fold volume reduction in the top phase as compared to that of the crude enzyme solution, and the isoamylase activity retention in the top phase was higher than 80%. With PEG systems of various molecular weights and concentrations, the partition coefficients of isoamylase tended to decrease with the increases of PEG molecular weight and concentration. These behaviors could be interpreted by the influence of hydrophobic interaction as well as free volume change.